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EP0807255B1 - Dispositif de dosage et de test pour l'immunoagregation amelioree par des liposomes - Google Patents

Dispositif de dosage et de test pour l'immunoagregation amelioree par des liposomes Download PDF

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Publication number
EP0807255B1
EP0807255B1 EP96909480A EP96909480A EP0807255B1 EP 0807255 B1 EP0807255 B1 EP 0807255B1 EP 96909480 A EP96909480 A EP 96909480A EP 96909480 A EP96909480 A EP 96909480A EP 0807255 B1 EP0807255 B1 EP 0807255B1
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Prior art keywords
analyte
liposomes
absorbent material
liposome
test
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German (de)
English (en)
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EP0807255A4 (fr
EP0807255A1 (fr
Inventor
Richard A. Durst
Matthew A. Roberts
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Cornell Research Foundation Inc
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Cornell Research Foundation Inc
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G01N33/586—Liposomes, microcapsules or cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G01N33/5438—Electrodes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/805—Test papers
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/81—Packaged device or kit
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/97—Test strip or test slide
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/80—Fluorescent dyes, e.g. rhodamine
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/805—Optical property
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/807—Apparatus included in process claim, e.g. physical support structures
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/807—Apparatus included in process claim, e.g. physical support structures
    • Y10S436/81—Tube, bottle, or dipstick
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/815—Test for named compound or class of compounds

Definitions

  • the present invention relates to a method for detecting or determining one or more analytes, and a test device used in the method. More particularly, the invention relates to a single-use test strip for use in an immunomigration assay employing marker-loaded liposomes for signal amplification.
  • PCBs Polychlorinated biphenyls
  • Aroclor trademark a trademark for industrial compounds used extensively as lubricants, fire retardants, immersion oils, dielectric and heat transfer fluids, as well as a multitude of other products.
  • Safe, S . Toxicology 1990, 3, 51-88. They have contaminated an enormous variety of media, primarily as a result of careless use, disposal, and accidents, and have now been identified by the EPA as priority pollutants to be targeted for remediation under the national Superfund program. Extensive efforts have recently been undertaken to characterize Superfund sites by both the EPA and various environmental remediation firms.
  • One of the chief obstacles to the prompt completion of such studies is the high cost and long turnaround time for conventional PCB analysis by off-site laboratories.
  • Immunoassays comprise one category of specific binding assays, which generally rely on the affinity of naturally occurring receptors or antibodies for specific compounds.
  • the specific binding pairs employed in immunoassays are either an antigen or a hapten, and the antibody produced in an immune response to the antigen or hapten.
  • Competitive immunoassays are generally based upon the competition between a specific analyte, the amount of which is to be determined, and a labelled form of the analyte or an appropriate analog thereof, which is used as an indicator, for a limited number of available binding sites on a binding material specific for the analyte.
  • the amount of analyte in the sample can be determined by measuring the amount of the unbound labelled analyte, which in some systems is physically separated from the bound indicator during the assay.
  • an assay can be completed without separating the bound and unbound indicator.
  • immunoassay label materials or markers include various enzymes, fluorescent dyes, chemiluminescent reactants, and radioisotopes. Such materials are often conjugated to the analyte, as in the case of enzymes and radioisotopes, or less frequently, carried within sacs such as animal erythrocytes, polymer microcapsules, or liposomes.
  • Immunoassays have been widely used for medical diagnosis for many years. More recently, immunoassays have been more broadly applied for the determination of toxic substances in the environment and in food. Practical applications for immunoassays in environmental analysis include evaluating the geographical scope and magnitude of pollutants, monitoring the fate and persistence of contaminants, and assessing the effectiveness of remediation efforts. Raw and processed foods must similarly be tested for chemical and biological contamination.
  • Immunoassay techniques have shown considerable promise for the characterization of PCB contamination.
  • Most assays have chosen the ELISA (enzyme-linked immunosorbent assay) format which is often based on the competition between sample analyte and analyte-enzyme conjugates for a limited number of antibody binding sites.
  • ELISA enzyme-linked immunosorbent assay
  • U.S. Patent No. 5,085,987 to Olson also describes an immunoassay employing a bibulous element such as a piece of paper affixed to plastic with adhesive.
  • the element is contacted with the test solution suspected of containing the analyte, to which has been added an antibody for the analyte and a conjugate of the analyte and a label.
  • the element contains a first receptor for the conjugate which is bound to a situs on the element separated from the contact portion, and a second receptor capable of binding the antibody for the analyte, which is bound to the element between the first receptor and the contact portion.
  • the test solution moves along the element by capillary action.
  • the situs is examined for the presence of conjugate, either by exposing the situs to a signal producing means capable of interacting with the label to produce a signal in a separate development step, such as an enzyme-catalyst-substrate system, or by directly measuring the signal from a radioactive label.
  • a signal producing means capable of interacting with the label to produce a signal in a separate development step, such as an enzyme-catalyst-substrate system, or by directly measuring the signal from a radioactive label.
  • U.S. Patent No. 4,939,098 to Suzuki, et al. discloses an immunoassay device for simultaneous determination of at least two components in a sample. At least two reagents, each of which reacts specifically with one of the components in the sample, are supported in optional places on a development layer. Residual components in the sample which do not react with the reagent first contacted by the sample continue to be moved past the place on the development layer where the first reagent is supported. After the movement of the unreacted components past each of the reagent places, the amount of the two reaction products still held in the development layer are measured. Test reagents may be included in liposomes, which are immobilized on the development layer by physical adsorption or chemical bonding.
  • a detectable label substance such as a chelating agent, an enzyme or a fluorescent substance may be enclosed in the liposomes in addition to the antibody or antigen test reagents for qualitative or quantitative analysis of sample components.
  • the liposomes or other label sacs are lysed by the antigen-antibody reaction or complement activity, to release label for detection or quantification.
  • Suzuki further describes an electric measurement method in which the liposomes contain a substance detectable with electrodes. A solution of the liposomes is removed from the development layer, and the amount of the component to be measured is quantified from the amount of signal produced at the electrode.
  • Suzuki's technique is not well-suited for use in the field, or for use by untrained personnel.
  • High voltage is required for the electrophoretic separation method, for example.
  • US Patent No 4,920,046 to Mac Farland et al (D2) relates to process, test device and test kit for a rapid assay having a visible readant.
  • Campbell discloses an immunoassay for determination of an analyte using a tracer, such as the analyte labelled with liposome-encapsulated markers.
  • the tracer can be visually determined without instrumentation and without further treatment of the tracer (such as sac lysing).
  • a binder for at least one of the analyte and the tracer is supported on a test area of a solid support, which is preferably nitrocellulose in the form of a card, test strip, or dipstick. Detection or quantification of the signal, e.g., color from a dye, is made in the test area of the device.
  • Competitive, sandwich, and inhibition embodiments of the assay are disclosed.
  • the Duracytes are coated with anti-fluorescein antibody and combined with antisera to five drugs (amphetamines, cannabinoids, cocaine metabolites, opiates, and PCP).
  • the test sample is added to this combination, and the entire mixture (test sample, Duracytes, and antisera) is loaded onto a multichambered vessel device.
  • the device automatically distributes the mixture into distinct assay channels, each containing different dried flourescein-drug conjugates.
  • Negative assays no drug present
  • form an agglutinated reaction product as a result of reactions between the Duracytes, the conjugate, and the anti-drug antibody
  • positive assays show no agglutination. Agglutination results in the production of characteristic banded patterns in the channels showing a negative result.
  • Parsons et al. thus requires two different antibodies for agglutination, one coated on the Duracytes and one immobilized on a solid surface.
  • Parsons relies on the production of signal for negative results, which is counter-intuitive, and its narrow dynamic range for detection effectively limits its usefulness when quantitation is desired. Also, the range of markers which can be applied to the Duracytes is limited.
  • the present invention relates to a test device for detecting or quantifying an analyte in a test sample.
  • the test device includes an absorbent material, having a contact portion at or proximate to one end.
  • a measurement portion is positioned away from the first end of the absorbent material.
  • the measurement portion has a receptor for a conjugate of an analog of the analyte to be determined and liposomes comprising a detectable marker.
  • the present invention further provides a method for detecting or quantifying an analyte in a test sample utilizing the test device of the invention.
  • the test sample is combined with a binding material specific for the analyte, and a conjugate of an analyte analog and liposomes comprising a detectable marker, to form a mixture.
  • the mixture is incubated for a time sufficient to allow competition between the conjugate and any analyte present for the binding material.
  • the mixture is then contacted with the contact portion at or proximate to one end of the absorbent material of the test device.
  • the test mixture is allowed to traverse the absorbent material, via capillary action, from the contact portion through a measurement portion of the absorbent material which is positioned away from the end to which the contact portion is at or proximate.
  • the measurement portion has a receptor for the analyte analog-liposome conjugate.
  • the device and method of the invention can be used directly in the field.
  • the device is used only once, and, therefore, is free from residual environmental contaminants other than what may be present in the sample to be measured. Samples can be assayed within minutes after collection, with the results immediately available on-site.
  • the device and method of the invention are much less complex than many of the prior.materials and methods.
  • a visible dye can be used as the detectable marker, eliminating the need for any detection or measurement instrumentation, and a separate marker or indicator development step is not required with any embodiment of the invention.
  • marker-loaded liposomes as used in the device and method of the invention provide a highly sensitive, rapid or even instantaneous signal production/amplification system.
  • the amount of marker measured in the measurement portion of the absorbent material of the test device is directly proportional to the analyte concentration in the sample.
  • the present invention is directed to a test device for detecting or quantifying an analyte in a test sample.
  • the test device includes an absorbent material which comprises a contact portion at or proximate to a first end of the absorbent material.
  • the absorbent material of the test device further comprises a measurement portion at a location on the absorbent material which is positioned away from the first end.
  • the measurement portion has a receptor for a conjugate of an analyte analog and liposomes, wherein the liposomes comprise a detectable marker.
  • the invention is also directed to a method for detecting or quantifying an analyte, including the steps of providing a test device as described immediately above, combining a binding material specific for the analyte with the conjugate and the test sample to form a mixture, incubating the mixture for a time sufficient to permit competition between the conjugate and the analyte for the binding material, contacting the mixture with the contact portion of the test device after incubation, allowing the mixture to migrate from the contact portion through the measurement portion of the absorbent material, detecting the presence amount of the marker in the measurement portion of the absorbent material and correlating the presence or amount of the marker in the measurement portion with the presence or amount, respectfully, of the analyte in the sample.
  • analyte is meant the compound or composition to be measured that is capable of binding specifically to an binding material.
  • binding material is meant a bioreceptor molecule such as an immunoglobulin or derivative or fragment thereof having an area on the surface or in a cavity which specifically binds to and is thereby defined as complementary with a particular spatial and polar organization of another molecule -- in this case, the analyte.
  • the binding material such as an antibody, can be monoclonal or polyclonal and can be prepared by techniques that are well known in the art such as immunization of a host and collection of sera or hybrid cell line technology.
  • the binding material may also be any naturally occurring or synthetic compound that specifically binds the analyte of interest.
  • IgG is a preferred binding material in accordance with the invention as it is bivalent and, therefore, tends to enhance the formation of aggregates of the conjugate and the binding material.
  • IgM may be particularly preferred for certain applications, as its 10 binding sites per molecule would be expected to promote the formation of large aggregates.
  • receptor any compound or composition capable of recognizing a particular spatial and polar organization of a molecule, e.g., an epitope or determinant site.
  • Suitable receptors in accordance with the invention include those capable of binding directly with the surface of the liposomes, or with a molecule bound on adhered to the surface of the liposomes.
  • an antibody specific for a liposome tag, sufficiently different in structure from the analyte of interest can be employed as a receptor for suitably derivatized liposomes.
  • Illustrative receptors include naturally occurring receptors, e.g., egg white avidin, streptavidin, thyroxine binding globulin, antibodies, Fab fragments, lectins, nucleic acids, protein A, protein G, and the like.
  • avidin or more preferably, anti-biotin antibody function as receptors for liposomes derivatized with biotin.
  • egg white aviden can be employed as the receptor, as it will bind directly to the liposome surface.
  • the method of the invention employs a conjugate of marker-encapsulating liposomes and an analyte analog.
  • Certain analytes of interest may be so intractable as to make direct conjugation with the liposome inconvenient, difficult, or even impossible. In such cases, it will be necessary to employ a reactive analog of the analyte of interest to prepare the conjugate.
  • analyte analog is meant either the analyte or an analog of the analyte which will react with or bind to the liposomes.
  • marker accumulating agent any ion, compound, or composition capable of trapping electroactive marker materials released from the liposome interiors. Ion-exchange resins are preferred marker accumulating agents in accordance with the invention.
  • the mixture formed by combining the binding material, the conjugate, and the test sample may be a solution, suspension, dispersion, or other mixture.
  • absorbent material is meant a porous material having a pore size of from 0.05 ⁇ m to 50 ⁇ m, preferably from 0.45 ⁇ m to 5 ⁇ m, which is susceptible to traversal by an aqueous medium in response to capillary force.
  • Such materials may be natural polymeric materials, particularly cellulosic materials, such as fiber-containing papers, e.g., filter paper, chromatographic paper, etc.; synthetic or modified naturally occurring polymers, such as nitrocellulose, cellulose acetate, poly(vinyl chloride), polyacrylamide, cross linked dextran, agarose, polyacrylate, activated nylon, etc.; either used by themselves or in conjunction with a support, as described below. Nitrocellulose is a preferred absorbent material.
  • the absorbent material may be polyfunctional or be capable of being polyfunctionalized to permit covalent bonding of receptors as well as to permit bonding of other compounds which form a part of the signal producing system.
  • the absorbent material which is employed in the test device and method of the invention is generally a cellulose ester with nitrocellulose giving exceptionally good results.
  • nitrocellulose refers to nitric acid esters of cellulose, which may be nitrocellulose alone, or a mixed ester of nitric acid and other acids, and in particular, aliphatic carboxylic acids having from one to seven carbon atoms, with acetic acid being preferred.
  • Such materials which are formed from cellulose esterified with nitric acid alone, or a mixture of nitric acid and another acid such as acetic acid, are often referred to as nitrocellulose paper.
  • nitrocellulose is a preferred material for producing the test device, it is to be understood that other materials, having a surface area sufficient for supporting the receptor and other agents to be immobilized thereon in a concentration as hereinbelow described, and a pore size suitable for accumulating aggregates formed from the conjugate and the binding material may also be employed for producing such test devices.
  • the absorbent material which is used in the device and method of the invention has a surface area such that is capable of supporting the receptor in an excess amount, i.e., in an amount sufficient to capture all of the conjugate which reaches the measurement zone during traversal of the absorbent material by the test mixture.
  • Absorbent materials having high surface areas are particularly preferred in that the receptor may be supported on such materials in a high concentration.
  • receptors, liposome lysing agents, and marker accumulating agents to the absorbent material may be accomplished by well-known techniques, for example, by spraying or spotting a solution of those materials onto the absorbent material.
  • the amount of receptor which is bound to the absorbent material at the measurement portion will vary depending upon the amount required to bind the unbound conjugate to enable an effective assay. Generally, the amount of receptor at the measurement portion will be at least 10 ⁇ g/cm 2 . However, the invention is not limited to a particular concentration of receptor on the absorbent material.
  • the receptor and members of the signal producing system can be bound to the absorbent material by adsorption, rather than covalent bonding, as long as such binding is non-diffusive. This will involve contacting the absorbent material with a solution containing the materials to be bound to the material and allowing the material to dry. In general, this procedure will be useful only where the absorbent material is relatively hydrophobic or has a high surface charge, and subsequent treatment with proteins, detergents, polysaccharides, or other materials capable of blocking nonspecific binding sites will be required.
  • the residual nonspecific' binding capacity of the absorbent material is saturated or blocked with one or more types of proteins or other compounds such as polyvinylpyrrolidone, polyvinylalcohol, other suitable polymeric blocking agents etc., which do not specifically bind the materials to be employed in the assay. Blocking is generally carried out after the receptor is applied to the strip, but it may be possible to block the strip before the receptor is applied depending on the particular receptor, blocking agent, and absorbent material employed.
  • the order of the steps of blocking the absorbent material and applying the liposome lysing agent and/or the marker accumulating agent may vary depending on the particular assay conditions and components employed.
  • the residual binding capacity of the substrate may be blocked so as to prevent nonspecific binding by the use of bovine serum albumin, as described in Towbin, et al., Proc. Nat'l. Acad. Sci., 76 (1979) 4350.
  • the techniques for preventing non-specific binding are generally known in the art, and such techniques are also generally applicable to preventing nonspecific binding in the assay of the present invention. Examples of particularly suitable techniques for blocking with polyvinylpyrrolidone and polyvinylalcohol are described, for example, Bartles, et al. Anal. Biochem., 140 (1984) 784, and in British Patent Specification GB 2204398 A, respectively.
  • a surfactant may be applied to the absorbent material in a concentration sufficient to promote homogeneous flow of the test solution across the test device, to facilitate migration of the analyte analog-liposome conjugate without lysis of the liposomes.
  • Suitable surfactants include BrijTM (polyoxyethylene ether), Tween 20TM (polyoxyethylenesorbitan monolaurate), Triton X-100TM (t-octylphenoxypolyethoxyethanol), sodium dodecylsulfate, n-octyl- ⁇ -D-glucopyranoside, Span 20TM, Nonindet P-40, ChapsoTM, TurgitolTM and sodium dioxycholate.
  • the concentration of the surfactant(s) employed in a blocking solution will depend, in part, upon the liposome composition.
  • surfactants may be incorporated in a concentration of from about 0 to about .01 volume percent of the blocking solution, preferably from about .001 to about .005 volume percent of the blocking solution. It is important that the concentration of surfactant applied to the absorbent material be controlled, as premature lysis of the liposomes may occur if the surfactant concentration is too high.
  • Tween 20TM is a preferred surfactant for use in a blocking solution.
  • polyvinylpyrrolidone assists in the accumulation of aggregates on the absorbent material, particularly for "Liposome-Antibody Aggregation"-type aggregates shown in Figure 9B, described more fully below.
  • the blocking agents block nonspecific binding sites on the absorbent material.
  • the blocking agents are selected from the group consisting of proteinaceous blocking reagents capable of inhibiting binding of molecules having a molecular weight of greater than about 1000 with said absorbent material and polymer blocking reagents capable of inhibiting binding of molecules having a molecular weight of less than about 1000 with said absorbent material.
  • the proteinaceous blocking reagent may be selected from the group consisting of gelatin, non-fat dry milk, bovine serum albumin, and keyhold limpet hemocyanin.
  • the polymer blocking reagent may be selected from the group consisting of polyvinylpyrrolidone and polyvinylalcohol
  • the surfactant may be selected from the group consisting of polyoxyethylene ethers, polyoxyethylenesorbitan monolaurate, t-octylphenoxypolyethoxyethanol, and sodium dodecylsulfate, octylglucopyranoside, and sodium dioxycholate.
  • the absorbent material can be a single structure such as a sheet cut into strips.
  • the absorbent material can be mounted on a support material.
  • the absorbent material may provide its own support.
  • the test device is a strip of particulate material bound to a support or solid surface such as found, for example, in thin-layer chromatography.
  • the absorbent material can be a sheet having lanes thereon, or be a uniform sheet capable of division into separate lanes by physical removal of the absorbent material from the support to induce lane formation, wherein a separate assay can be performed in each lane as shown in Figures 1-3 and 6-7.
  • the absorbent material can have a shape that is rectangular, circular, oval, trigonal, or the like, provided that there is at least one direction of traversal of a test mixture by capillary migration. Other directions of traversal may occur such as in an oval or circular piece contacted in the center with the test mixture. However, the main consideration is that there be one direction of flow from the contact portion through the measurement portion. In this discussion strips of absorbent material are described by way of illustration and not limitation.
  • the absorbent material of the test devices in accordance with the invention preferably comprises a region for accumulation of aggregates formed from the conjugate and the binding material, as described in more detail, below.
  • this region for accumulation is positioned away from the liposome lysing agent, and either between the liposome lysing agent and the contact portion, or in the contact portion.
  • this region for accumulation is positioned away from the measurement portion, and either between the measurement portion and the contact portion, or in the contact portion.
  • the measurement portion In constructing the test devices in accordance with the invention, it is desirable to position the measurement portion as close as possible to the contact portion in order to minimize the time necessary for the test mixture to reach and pass through the measurement portion. However, it is important that the measurement portion and contact portion not be so close as to contact one another, and to avoid having the test mixture come in contact with the measurement portion other than by capillary transport of the test mixture through the measurement portion. In other words, the measurement and contact portions should be separated sufficiently to avoid premature or unwanted contamination of the measurement portion through human error in manipulating the device. When there are multiple measurement portions positioned on the absorbent material (as described below for multi-analyte testing) the individual measurement zones may be close to one another and may, in certain cases, even overlap.
  • Figure 1 is a schematic of a test device in accordance with the invention, depicted immediately after insertion into control mixture 209 and test mixture 208, which are held in tray 210 having partition 216 extending across the entire width of tray 210 to divide tray 210 into separate compartments for the control and test mixtures.
  • absorbent material 212 is mounted on support 214.
  • the test device shown in Figure 1 is divided into two channels, namely, control channel D and test channel E, with measurement portions 206.
  • Control channel D includes measurement portion 206d, which, as described above, has a receptor for the appropriate analyte analog-liposome conjugate bound thereto.
  • Test channel E similarly has measurement portion 206e, which has been constructed to recognize and bind the analyte analog-liposome conjugate, respectively, as described above.
  • each channel of the test strip is the end of the strip to be inserted into the test or control mixtures.
  • Test mixture 208 is typically prepared, as described below, by combining a sample known or suspected to contain the analyte with the analyte analog-liposome conjugate and a binding material specific for the analyte in an aqueous medium.
  • control mixture 209 is typically prepared to have the same concentration of the conjugate as test mixture 208, the same concentration of binding material as test mixture 208 and a known concentration of analyte.
  • the mixture containing the binding material, the conjugate, and the analyte (if present) is then incubated for a time sufficient to permit the conjugate and the analyte to compete with one another for binding with the binding material.
  • the incubation time will vary with the particular assay, however, in most cases, from about less than 1 minute to about 30 minutes will be sufficient to allow the competition reaction to reach or approach completion. Incubation times of from about 1 minute to about 30 minutes are easily achieved with the method of the invention, and are preferred, as one of the significant advantages of the present invention is the speed with which testing for analytes can be carried out.
  • control solution 209 the contact portion of absorbent material 212 of control channel D is inserted into control solution 209, while the contact portion of absorbent material 212 of test channel E is inserted into test solution 208.
  • Wetting of absorbent material 212 by capillary action is allowed to continue at least until measurement portions 206d and 206e are wet, (and preferably, until the solvent front reaches the end of the absorbent material) with control solution 209 and test solution 208, respectively.
  • Control solution 209 and test solution 208 continue to traverse channels D and E of the test device into and through measurement portions 206d and 206e, where the conjugate is trapped and accumulated in measurement portions 206d and 206e by the specific conjugate receptor bound thereto.
  • qualitative measurement of the marker in measurement zones 206d and 206e may be made visually or instrumentally when the marker is a visible dye.
  • the intensity of the color in measurement portions 206d and 206e may be visually compared with a series of reference standards, such as in a color chart, for a semi-quantitative determination of the amount of analyte in the sample.
  • other types of markers as described below, may be detected and measured using instrumentation such as a reflectometer, spectrophotometer or fluorimeter.
  • test device in accordance with the invention is shown schematically in Figure 2.
  • the test device is shown before insertion into sample compartment 110 containing a wicking reagent such as carrier solution 108, which is generally a buffered saline solution.
  • carrier solution 108 which is generally a buffered saline solution.
  • the test device is divided into high control channel A, test channel B, and low control channel C.
  • absorbent material 112 is supported on support 114.
  • the test device includes wicking portions 100, contact portions 102, and measurement portions 106.
  • High control channel A includes contact portion 102a, and measurement portion 106a.
  • Test channel B similarly includes contact portion 102b, and measurement portion 106b.
  • low control channel C includes contact portion 102c, and measurement portion 106c.
  • test device shown schematically in Figure 2 is designed for the simultaneous measurement of the analyte in a test sample and high- and low-level control compositions to provide linear interpolation and verification of response.
  • a high-level control solution or mixture, the test mixture prepared and incubated as described above in connection with Figure 1, and a low-level control solution or mixture are spotted onto contact portions 102a, 102b, and 102c, respectively, prior to insertion of the test device into carrier solution 108.
  • the support for the absorbent material where a support is desired or necessary will normally be hydrophobic, water insoluble, non-porous, and rigid, and usually will be of the same length and width as the absorbent strip but may be larger or smaller.
  • a wide variety of organic and inorganic materials, both natural and synthetic, and combinations thereof, may be employed, provided only that the support does not interfere with the production of signal from the marker.
  • Illustrative polymers include polyethylene, polypropylene, poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethylene terephthalate), nylon, poly(vinyl chloride) poly(vinyl butyrate), glass, ceramics, metals, and the like.
  • the size of the piece of absorbent material is dependent on several considerations.
  • the primary consideration, as described further below, is to capture unaggregated conjugate at the measurement portion to give a sufficient signal so that a sensitive and accurate assay is achieved.
  • the following discussion is primarily focused on strips of absorbent material for purpose of illustration and not limitation. As mentioned above, other shapes such as circular, oval, trigonal, and the like, fall equally within the scope of this invention. The dimensions thereof and other parameters can be determined by those skilled in the art with reference to the disclosure herein.
  • the length and thickness of the strip control the amount of mixture that can pass through the measurement portion. If the transfer of a large volume of test mixture is desired, the fluid capacity of the strip above the measurement portion must be sufficient to accommodate the desired volume.
  • an absorbing pad or sponge may be used to contact the end of the strip opposite the end used to contact the test mixture. An absorbing pad or sponge may be used in this manner in situations when it is desirable to pull a larger volume of the test mixture across the test device so as for example, to increase the concentration of the conjugate in the measurement zone.
  • the width of the strip will generally be relatively narrow, usually less than 20 mm preferably less than 10 mm. Generally, the width of the strip will not be less than about 2 mm and will usually range from about 2 mm to 10 mm, preferably from about 3 mm to 6 mm.
  • the test device in accordance with the invention may be modified for simultaneous multiple analyte detection or determination.
  • the length of the strip will depend on the concentration of the analyte and practical considerations such as ease of handling and the number of measurement portions on the strip and will be about 4 cm to 20 cm, usually about 5 cm to 15 cm, preferably about 6 to 13 cm but may be of any practical length.
  • the structure of the strip can be varied widely and includes fine, medium fine, medium, medium coarse and coarse. In general, smaller pore size and finer material will provide slow capillary flow and more efficient capture of unaggregated conjugate on the strip. More significantly, an absorbent material having a smaller pore size will trap smaller aggregates. Courser, more porous materials provide faster flow, but the efficiency of capture is reduced. Selection of the porosity of the material depends on the rate of binding of the components for a given assay.
  • the position of the measurement portion should be governed by the basic principle involved in the present invention.
  • the measurement portion should be at least 3 mm, preferably at least 8 mm, from the contact portion of the strip.
  • the measurement portion should be positioned on the absorbent material so as to enable the test mixture to pass through the measurement portion by capillary action so as to capture the unaggregated conjugate. Where several measurement portions are used for multi-analyte determinations, the measurement portions can be grouped close together or apart. Depending upon the signal production and detection system employed, it may be necessary in some cases to separate the portions so as to avoid compromising the resolution of the signals.
  • the protocol will normally involve combining the sample suspected of containing the analyte with the binding material and conjugate in an aqueous medium to form an aqueous test mixture or solution.
  • the sample may be derived from a wide variety of sources, such as physiologic fluids, illustrated by saliva, sweat, serum, plasma, urine, tear fluid, spinal fluid, etc., chemical processing streams, food, waste water, natural waters, soil extracts, etc.
  • Various addenda may be added to adjust the properties of the test mixture, or of a carrier solution used as a wicking reagent, depending upon the properties of the other components of the device, as well as on those of the liposomes or the analyte analog-liposome conjugate, or the analyte itself.
  • solution addenda which may be incorporated into test, control, or carrier solutions or mixtures in accordance with the invention include buffers, for example, pH and ionic strength, and sample or analyte solubilizing agents, such as, for example, nonpolar solvents.
  • test sample saliva sample
  • binding material containing the analyte
  • conjugate conjugate to one another is not critical. Generally, however, it is preferred to allow the binding material and test sample to interact briefly before the addition of the conjugate to compensate for the competitive advantage enjoyed by the conjugate with its multiple binding material binding sites.
  • the contact portion of the absorbent material which usually includes the end of the absorbent material to which the contact portion is proximate, is contacted with test mixture, usually by immersion of the contact portion into the test mixture. Wetting of the absorbent material by capillary action is allowed to continue at least until the measurement portion is wet, preferably until the solvent front reaches the end of the absorbent material opposite the first end.
  • the test mixture may be contacted with the absorbent material by spotting the test mixture (following incubation to form aggregates) onto the absorbent material in the contact portion.
  • the contact portion includes a wicking portion at the first end of the absorbent material. In use, the wicking portion of the contact portion is inserted into a wicking reagent after the test mixture is spotted onto the contact portion, outside of the wicking portion.
  • the test mixture For the most part, relatively short times are involved for the test mixture to traverse the strip. Usually, traversal of the test mixture over the strip will take at least 30 seconds and not more than 1/2 hour, more usually from about 1 minute to 10 minutes. In accordance with the method of the invention, the signal is rapidly, even immediately, detectable.
  • the conjugate of the analyte analog and the marker-encapsulating liposome may be prepared by procedures generally known in the art, with the particular procedure used in a given case being dependent upon the analyte or analog which is employed. Such techniques include covalent coupling, derivatization or activation, and the like.
  • the liposomes may be produced from a component which has been derivatized with the analyte, whereby the liposomes, when produced, are conjugated with the analyte.
  • the liposomes, including the marker may be initially formed, followed by conjugating the liposomes with the analyte or analyte analog by procedures known in the art.
  • Liposomes can be prepared from a wide variety of lipids, including phospholipids, glycolipids, steroids, relatively long chain alkyl esters; e.g., alkyl phosphates, fatty acid esters; e.g. lecithin, fatty amines, and the like.
  • a mixture of fatty materials may be employed, such as a combination of neutral steroid, a charge amphiphile and a phospholipid.
  • Illustrative examples of phospholipids include lecithin, sphingomyelin, and dipalmitoylphosphatidylcholine, etc.
  • Representative steroids include cholesterol, chlorestanol, lanosterol, and the like.
  • Representative charge amphiphilic compounds generally contain from 12 to 30 carbon atoms.
  • Mono- or dialkyl phosphate esters, or alkylamines; e.g. dicetyl phosphate, stearyl amine, hexadecyl amine, dilaurylphosphate, and the like are representative.
  • the liposome sacs are prepared in aqueous solution containing the marker whereby the sacs will include the marker in their interiors.
  • the liposome sacs may be prepared by vigorous agitation in the solution, followed by removal of the unencapsulated marker. Further details with respect to the preparation of liposomes are set forth in U.S. Patent No. 4,342,826 and PCT International Publication No. WO 80/01515.
  • the signal producing system includes a marker included in the interior of the conjugated liposomes.
  • Suitable markers include fluorescent dyes, visible dyes, bio- and chemiluminescent materials, enzymatic substrates, and radioactive materials. Visible dyes and radioactive materials can be measured without lysis of the liposomes. However, even when liposome lysis is required, as when the other marker materials are used, a separate lysing step is not necessary, because a liposome lysing agent may be non-diffusively bound directly to the absorbent material as, for example, in the measurement zone.
  • Suitable liposome lysing materials include surfactants such as octylglucopyranoside, sodium dioxycholate, sodium dodecylsulfate, polyoxyethylenesorbitan monolaurate sold by Sigma under the trademark Tween-20, and a non-ionic surfactant sold by Sigma under the trademark Triton X-100, which is t-octylphenoxypolyethoxyethanol.
  • Octylglucopyranoside is a preferred lysing agent for many assays, because it lyses liposomes rapidly and does not appear to interfere with signal measurement.
  • complement lysis of liposomes may be employed.
  • a qualitative or semi-quantitative measurement of the presence or amount of an analyte of interest may be made with the unaided eye when visible dyes are used as the marker.
  • the intensity of the marker may be measured directly on the absorbent material using a quantitative instrument such as a reflectometer, fluorimeter, spectrophotometer, electroanalyzer etc.
  • a marker which is visible under the assay conditions is used so that the presence and/or amount of analyte may be determined without further treatment and without the use of instrumentation, e.g., by use of a liposome containing a dye as the marker.
  • a conjugate of an analyte analog and marker-loaded liposome are combined in an aqueous medium with a sample suspected of containing the analyte and a binding material specific for the analyte, to provide an aqueous test mixture.
  • the liposomes incorporated in the conjugate have multiple analyte analog molecules bound hereto and, therefore, multiple binding sites for the binding material.
  • binding material will react exclusively with the conjugate, resulting in the formation of relatively large aggregates, each of which may include multiple liposomes (see, for example, the two examples of conjugate-binding material aggregates in Figure 9B).
  • the large aggregates formed during the incubation will tend to be retained in the interstices of the nitrocellulose matrix and will form an "aggregation zone" on the absorbent material, usually at or near the meniscus of the test mixture when the device is inserted as shown in Figure 1 into the test mixture.
  • the analyte inhibits conjugate aggregation.
  • the greater the concentration of analyte in the test sample fewer aggregates will form and those that do form will be relatively limited in size. Smaller particles, including unaggregated liposome-analyte analog conjugate, will not be retained at the "aggregation zone" and will continue to migrate until bound in the measurement zone.
  • the conjugates that do not aggregate will be proportional to the amount of analyte in the mixture, and will bind to the measurement portion.
  • a receptor capable of binding to the conjugate is preferably non-diffusively bound to the absorbent material at the measurement portion.
  • the contact portion of the absorbent material is contacted with the test mixture, which will traverse the absorbent material through capillary action. This transversal can be upward, downward, horizontal or combinations thereof.
  • the amount of the conjugate that becomes bound to the measurement portion through binding to the receptor is related to the amount of analyte in the sample.
  • the signal-producing system provides a detectable signal at the measurement portion only when the conjugate is bound to the receptor in the measurement portion, so that the presence of the analyte may be determined by detecting the signal at the measurement portion. Binding of the conjugate to the receptor may occur directly to a binding site on the liposome.
  • the present invention provides for an immunoseparation of aggregated conjugate from unaggregated conjugate. This is accomplished as a result of the inability of aggregated conjugate to proceed beyond a certain position on the absorbent material.
  • the movement of the test mixture along the absorbent material is due to capillary action. This capillary movement along the absorbent material causes the test mixture to be carried to and through the measurement portion.
  • Measurement of the marker-loaded liposomes takes place in the measurement portion of the absorbent material.
  • concentration or accumulation of the conjugate may be achieved by various immunospecific binding reactions as described above.
  • the conjugate of the analyte analog and the liposomes is further conjugated to biotin.
  • the assay is carried out in the same way but receptor for biotin is used, such as avidin or antibody specific for biotin.
  • biotinylated conjugate reaches the measurement portion and is bound by the anti-biotin or avidin.
  • avidin avidin
  • an avidin collection zone provides a simple solution to give the desired direct readout measurement.
  • the solvent for the test solution will normally be an aqueous medium, which may be up to about 40 weight percent of other polar solvents, particularly solvents having from 1 to 6, more usually of from 1 to 4, carbon atoms, including alcohols, dimethylformamide and dimethylsulfoxide, dioxane and the like.
  • the cosolvents will be present in less than about 20 weight percent. Under some circumstances, depending on the nature of the sample, some or all of the aqueous medium could be provided by the sample itself.
  • the pH for the medium will usually be in the range of 4-10, usually 5-9, and preferably in the range of about 6-8.
  • the pH is chosen to maintain a significant level of binding affinity of the binding members and optimal generation of signal by the signal producing system.
  • Various buffers may be used to achieve the desired pH and maintain the pH during the assay.
  • Illustrative buffers include borate, phosphate, carbonate, tris, barbital and the like.
  • the particular buffer employed is usually not critical, but in individual assays, one buffer may be preferred over another.
  • the concentration of electrolytes in the medium will usually be adjusted to achieve isotonicity or equi-osmolality with the solution in the interior of the liposomes to prevent their crenation or swelling.
  • temperatures are normally employed for carrying out the assay.
  • the temperatures for the assay and production of a detectable signal will generally be in the range of about 4-45°C., more usually in the range of about 10-38°C., and frequently, will be ambient temperatures, that is, about 15-25°C.
  • the concentration, in the liquid sample, of analyte which may be assayed will generally vary about 10 -3 to about 10 -15 M, more usually from about 10 -5 to 10 -10 M. Considerations such as the concentration of the analyte of interest and the protocol will normally determine the concentration of the other reagents.
  • test device may also assay a test solution for a plurality of analytes such as toxic chemicals, or screen for one or more of a plurality of analytes.
  • the test device includes multiple measurement portions, each of which has a distinctive receptor specific for one of several conjugatable ligands, which are chosen, in part, so as not to interfere with any of the analytes of interest.
  • the test solution, and control solution are formed by mixing together in an aqueous medium the sample and a plurality of liposome conjugates each of which comprises (a) an analog for one of the analytes and (b) a ligand which will bind specifically to one of the receptors in one of the measurement portions of the device.
  • the strip contains a separate measurement portion for each analyte.
  • a mixture of binding materials specific for each of the analytes is then employed in the test solution.
  • the conjugate of each of the analytes to be determined in this embodiment of the invention may include a marker which is detectable distinctly from the other markers. With different encapsulated dyes, the results of the assay can be "color coded".
  • each analyte may be determined by assignment of each conjugate/analyte to its own measurement portion for concentration and measurement.
  • the measurement portion has bound thereto separate receptors capable of binding different analyte analog-liposome conjugates through the recognition of the receptors for separate ligands on the different analyte analog-liposome conjugates.
  • a screening assay it is possible to conduct a screening assay to determine, for example, whether any of a group of analytes is present in the sample.
  • the liposomes attached to each analyte analog can have a different dye encapsulated, and a multi-wavelength detector can be used in a measurement portion, such as an egg-white avidin portion.
  • the present device can be provided in a kit in packaged combination with predetermined amounts of reagents for use in assaying for an analyte or a plurality of analytes.
  • reagents for use in assaying for an analyte or a plurality of analytes.
  • other additives such as ancillary reagents may be included, for example, stabilizers, buffers, and the like.
  • the relative amounts of the various reagents may be varied widely, to provide for concentration in solution of the reagents which substantially optimizes the sensitivity of the assay.
  • the reagents can be provided as dry powders, usually lyophilized, including excipients, which on dissolution will provide for a reagent solution having the appropriate concentrations for performing the assay.
  • the kit or package may include other components such as standards of the analyte or analytes (analyte samples having known concentrations of the analyte).
  • FIG 3 is a schematic of a cross-section of a commercially useful test device in accordance with the invention.
  • Absorbent material 420 is supported on strip holder 410, which may be composed of any inert rigid or semi-rigid support material and is preferably composed of plastic.
  • absorbent material 420 includes contact portion 402, and measurement portion 424, having a receptor for a conjugate of an analyte analog and marker-encapsulating liposomes bound thereto.
  • the absorbent material of the test device also includes end-point indicator portion 408, as described below. Compartment covers 414 and 418 provide further support and protection for absorbent material 420.
  • Covers 414 and 418 which are constructed of the same or similar rigid or semi-rigid support materials as strip holder 410, may, with strip holder 410, comprise a single molded piece. Alternatively, covers 414 and 418 may be prepared from a transparent material which allows viewing of absorbent material 420, and may be connected to strip holder 410.
  • the device shown in Figure 3 further include windows 416 which provide visual access to measurement portion 424 and end-point indicator portion 408 of the absorbent material.
  • Windows 416 may be made from a transparent material such as plastic or glass. Alternatively, they may be prepared from an opaque material such as a colored plastic incorporating holes through which absorbent material 420 may be seen.
  • Air vent 426 provides an outlet for air forced out of absorbent material 420 as a test solution or control solution migrates along absorbent material 420 from contact portion 402 to end-point indicator portion 408.
  • test solution containing the appropriate analyte analog-liposome conjugate, a binding material specific for the analyte, and known or suspected to contain the analyte, is incubated as described above, and then is spotted or dropped onto contact portion 402 of absorbent material 420.
  • Contact portion 402 is wet with the test or control solution, or a carrier solution or wicking reagent after initial application of the test or control solution, until the solution traverses the absorbent material 420 from contact portion 402 to end-point indicator portion 408.
  • Conjugate which is not aggregated and accumulated at or near the contact portion is accumulated in measurement portion 424 as a result of the binding reaction between the conjugate and the receptor bound to measurement portion 424.
  • the signal from the marker may be visually read through window 416 over measurement portion 424.
  • the signal from accumulated liposome-encapsulated marker may be instrumentally read by, for example, a spectrophotometer which is adapted for use with the device in accordance with the invention.
  • a layer of a material which will gradually dissolve upon contact with the test mixture may be applied to absorbent material 420 over contact portion 402.
  • the test sample, conjugate, and binding material can then be combined and poured into the sample well of the device shown in Figure 3, onto the dissolving layer, or the test mixture components may be introduced individually into the well.
  • the dissolving layer thus serves as a barrier between the test mixture and contact portion 402 during incubation. During incubation, the layer gradually dissolves, and the test mixture contacts contact portion 402.
  • the dissolving layer may be formed from any suitable material which will not interfere with the assay and which will gradually dissolve upon contact with the test mixture, such as, for example, hard-caked sucrose on cellulose membrane materials.
  • Figure 5 is a side-by-side schematic comparison of test devices substantially as described in copending application Serial Number 08/135,741 (Figure 5A) and test devices in accordance with the invention (Figure 5B). In each of Figures 5A and 5B, the devices are depicted with increasing test sample concentrations of PCBs from left to right.
  • test devices shown in Figure 5A have competitive binding portions 804 (from left to right 804c, 804d, and 804e, respectively), and measurement portions 806 (from left to right, 806c, 806d, 806e, respectively), on absorbent material 812.
  • the devices shown in Figure 8B have measurement portions 860 (from left to right, 860f, 860g, and 860h, respectively), on absorbent material 880.
  • Figure 5B illustrates test devices used in accordance with the method of the invention (i.e., the steps of combining the binding material, test sample, and conjugate to form a mixture, incubating the mixture to allow competition, and allowing the mixture to traverse the absorbent material from the contact portion through the measurement portion).
  • Figure 5B includes regions for accumulation of aggregates formed from the conjugate and the binding material ("aggregation zones") 840 (from left to right, 840f, 840g, and 840h), illustrating where accumulation of aggregates in accordance with the invention has occurred relative to the position of the measurement portion on the strip.
  • aggregation zones regions for accumulation of aggregates formed from the conjugate and the binding material
  • portions 806 in Figure 8A and 860 in Figure 5B are analogous in that they are each measurement or “collection” portions and have, therefore, been designated in Figure 5 as C1 and C2, respectively.
  • Portions 804 in Figure 5A and 840 in Figure 5B have been designated R1 and R2, respectively, to indicate their relationship with the competitive binding reactions which occur in the methods described in copending application Serial Number 08/135,741 (depicted in Figure 5A) and the present invention (depicted in Figure 5B).
  • the present invention is applicable to procedures and products for determining a wide variety of analytes.
  • analytes there may be mentioned: environmental and food contaminants, including pesticides and toxic industrial chemicals; drugs, including therapeutic drugs and drugs of abuse; hormones., vitamins, proteins, including antibodies of all classes; peptides; steroids; bacteria; fungi; viruses; parasites; components or products of bacteria, fungi, viruses, or parasites; allergens of all types; products or components of normal or malignant cells; etc.
  • T 4 T 3 ; digoxin; hCG; insulin; theophylline; leutinizing hormones and organisms causing or associated with various disease states, such as streptococcus pyrogenes (group A), Herpes Simplex I and II, cytomegalovirus, chlamydiae, etc.
  • group A streptococcus pyrogenes
  • Herpes Simplex I and II Herpes Simplex I and II
  • cytomegalovirus chlamydiae, etc.
  • the invention may also be used to determine relative antibody affinities.
  • the assay may be qualitative (presence or absence of certain level of analyte) or quantitative or semi-quantitative.
  • suitable standards and/or standard curves the term "standard curve” is used in a generic sense to include a color chart) is deemed to be within the scope of those skilled in the art from the teachings herein.
  • Examples 1 and 2 illustrate preparation and use of a test device substantially as shown and described in co-pending application Serial No. 08/135,741. , The device and methods described in Examples 1 and 2 are hereinafter referred to as the "liposome immunocompetition” or "LIC" format for convenience.
  • Bovine serum albumin BSA
  • N-succinimidyl-S-acetylthioacetate SATA
  • dipalmitoyl phosphatidyl ethanolamine DPPE
  • cholesterol PVP
  • PVP poly(vinylpyrrolidone)
  • Tween-20 triethylamine
  • Molybdenum Blue spray reagent Isosulfan Blue and Sephadex G-50 were purchased from Sigma (St. Louis, MO).
  • Dipalmitoyl phosphatidyl choline (DPPC) and dipalmitoyl phosphatidyl glycerol (DDPG) were obtained from Avanti Polar Lipids (Alabaster, AL). Sulforhodamine B was purchased from Eastman (Rochester, NY), and Fast Green FCF from Allied Chemical (New York, NY). Carnation non-fat dry milk powder (CNDM) was obtained locally. Protein assay dye reagent, goat anti-rabbit IgG alkaline phosphatase conjugate and the substrates for alkaline phosphatase were purchased from Bio-Rad (Hercules, CA). Egg white avidin was obtained from Molecular Probes (Eugene, OR).
  • a protein-binding membrane (absorbent material) with a plastic backing to provide rigidity was required, and nitrocellulose membrane supported in this manner was found to be the most suitable.
  • An airbrush was used to dispense the antibody and egg white avidin solutions for immobilization.
  • the membrane was cut to a desired size (7.9 cm high and a suitable width for later subdivision into strips 5 mm wide), thoroughly wetted with 10% methanolic TBS (tris buffered saline, pH 7.0) and dried before application of antibody and avidin solutions.
  • TBS tris buffered saline, pH 7.0
  • the membrane sheet was mounted on a mobile platform that moved at a constant rate in front of the airbrush used to spray the antibody solution at a concentration between 0.2 and 1 mg ml -1 (depending on preparation) onto the competitive binding portion of the membrane, and egg white avidin solution at 1 mg ml -1 onto the measurement portion of the membrane.
  • the protein bands were allowed to vacuum dry for 1 hour.
  • the coated nitrocellulose sheet was then immersed in blocking agent (a solution of 2% polyvinylpyrrolidone and 0.002% Tween-20 in TBS) for 1 hour on a rotating shake and dried under vacuum for 3-4 hours.
  • blocking agent a solution of 2% polyvinylpyrrolidone and 0.002% Tween-20 in TBS
  • Prepared sheets were stored at 4°C in the presence of silica gel desiccant until ready for use.
  • the sheets were cut into strips using a paper cutter when required.
  • the final strips were 5 x 79 mm with a 5 mm long antibody zone 15 mm above the bottom of the strip and a similar egg white avidin zone 35 mm from the bottom.
  • a thiolating reagent, SATA described in Duncan, et al., Anal. Biochem., 132 (1983) 68
  • SATA thiolating reagent
  • Twenty mg of DPPE were suspended in 3 ml of 0.7% triethylamine in chloroform and sonicated under nitrogen for 1 minute in a 45°C. bath.
  • To the DPPE 2 molar equivalents of SATA in 1 ml of the same solvent were added slowly. The reaction flask was capped and stirred at room temperature for ca.
  • the reaction mixture was stirred at 45°C for 2 hours, with the pH being maintained at 8.2, and the reaction was allowed to continue at 45°C overnight (ca. 17 hours).
  • the product was purified on a preparatory silica gel plate using the solvent system chloroform-acetone-methanol-glacial acetic acid-water (60:20:20:5:4, v/v).
  • the purified Alachlor-DPPE conjugate was quantified by Bartlett's phosphorous assay described in Bartlett, J. Biol. Chem., 234 (1959) 466.
  • TLC plates was dried, blocked for 1 hour in a solution of 1% BSA and 0.5% CNDM in TBS, washed three times for 10 minutes each in TBST (TBS containing 0.05% Tween-20), and placed overnight in a solution containing the antibody to Alachlor (20 pg/ml in TBST). The plate was washed three times for 10 min each in TBST and placed in a solution containing a goat anti-rabbit alkaline phosphatase conjugate (stock diluted 1:3000 with TBST containing 0.02% BSA) for 2 hours.
  • TBST TBS containing 0.05% Tween-20
  • the plate was washed as before and developed with the substrate for alkaline phosphatase (nitroblue tetrazolium in aqueous DMF with magnesium chloride and 5-bromo-4-chloro-3-indolyl phosphate in DMF, prepared according to the manufacturer's instructions).
  • alkaline phosphatase nitrogen blue tetrazolium in aqueous DMF with magnesium chloride and 5-bromo-4-chloro-3-indolyl phosphate in DMF, prepared according to the manufacturer's instructions.
  • color development was complete (10 min) the plate was washed in distilled water and dried.
  • a purple spot indicated the presence of Alachlor.
  • the other TLC plate was sprayed with molybdenum blue reagent (1.3% molybdenum oxide in 4.2 M sulfuric acid) which is specific for phospholipids.
  • the Alachlor-DPPE spot appeared purple with the alkaline phosphatase substrate stain and blue with the molybdenum
  • Liposomes were formed by the reversed-phase evaporation method, as described in Szoka, et al., Biochim. Biophys. Acta, 601 (1980) 559, and O'Connell, et al., Anal. Chem., 31 (1985) 142, from a mixture of DPPC, cholesterol, DPPG, and Alachlor-DPPE conjugate in a molar ration of 5:5:0.5:0.01. Forty-three ⁇ mol of this mixture were dissolved in 4.2 ml of a solvent mixture containing chloroform-isopropyl ether-methanol (6:6:1, v/v).
  • the diameters of the liposome preparations were measured by laser scattering in a LA-900 particle size distribution analyzer (Horiba, Irvine, CA), using the manufacturers method, except that the usual sonication step was omitted to avoid lysis (rupture) of the liposomes. Finally, to remove any unencapsulated dye, the liposomes were gel filtered on a 1 x 14 cm Sephadex G-50 column and dialyzed overnight against TBS at 4°C. When stored at 4°C, there was no significant leakage of dye over a period of 9 months as described below.
  • Sulforhodamine B was chosen as the dye for encapsulation, as described in O'Connell, above, and Chen, et al., Anal. Biochem. , 172 (1988) 61, because of its fluorescence and high visible extinction coefficient.
  • 20 mM Tris was used to buffer the dye solution. The pH was adjusted to 7.0 with NaOH to effect dissolution. The final solution contained 100 mM dye in 20 mM Tris at a pH of 7.0 with an osmolarity approximately equal to TBS, which was the buffer routinely used in all aqueous operations of the experiments. In some experiments 200 mM Sulforhodamine B was used to give a greater color intensity on the strips.
  • non-fluorescent dyes Isosulfan Blue and Fast Green FCF were encapsulated by the same methods as Sulforhodamine B.
  • Liposomes were prepared by the reversed-phase evaporation method described in Szoka and O'Connell, above, but without extrusion through polycarbonate filters, giving a high yield of liposomes. However, these heterogeneously sized liposomes did not migrate evenly on the test strips used in the assay. This was improved by passing the preparations sequentially twice through each of two polycarbonate filters of 1.0 and 0.4 ⁇ m nominal pore diameter. Liposomes passed only through the 1.0 ⁇ m filter had a mean diameter of 1.82 ⁇ m, with a standard deviation of 0.8, while those passed through both filters had a mean diameter of 0.68 ⁇ m with a standard deviation of 0.12.
  • the liposomes were stored at 4°C., and the temporal stability was studied over time by measuring the percentage of free dye in the preparation, thereby allowing calculation of the percentage of the liposomes that had lysed.
  • the assay device configuration consists of a wicking reagent containing Alachlor-tagged liposomes and a test strip comprised of a wick, an immobilized anti-Alachlor zone and an egg white avidin capture zone in sequence.
  • the assay is performed by dispensing 100 ⁇ l (2 drops) of the sample or control solution and 50 ⁇ l (1 drop) of a three times concentrated TBS buffer into a 10 x 75 mm glass test tube, mixing the contents, and adding 50 ⁇ l (1 drop) of a liposome solution (stock liposome solution diluted 1:50, dilution varying according to preparation).
  • test tube is shaken mildly to mix the contents and the test strip is inserted into the tube; the strip is left in the tube until the solution front reaches the end of the strip (about 8 min); the strip is removed and air dried.
  • the color intensity of the antibody zone and the egg white avidin zone are estimated either visually or by scanning densitometry, as described in Reeves, (1993).
  • the measurement of the extent of the competitive binding reactions of the analyte molecules and the tagged liposomes to the immobilized antibodies was optical. Visual estimation of the color intensity can be used, but for more accurate quantitation during development it was found to be preferable to use a computer scanner and Scan Analysis densitometry software (Biosoft, Ferguson, MO) to convert the red coloration into greyscale readings that can be measured.
  • Examples 3-9 illustrate the method of the invention, and preparation and use of the test devices in accordance with the invention, hereinafter referred to as the "liposome immunoaggregation" or -"LIA" format for convenience.
  • the LIA and LIC format are also illustrated together in the following examples.
  • Dipalmitoyl phosphatidyl ethanolamine DPPE
  • cholesterol Tween-20
  • triethylamine tris(hydroxymethyl)aminomethane
  • Tris tris(hydroxymethyl)aminomethane
  • Dipalmitoyl phosphatidyl choline DPPC
  • dipalmitoyl phosphatidyl glycerol DPPG
  • Avanti Polar Lipids, Inc. Avanti Polar Lipids, Inc. (Alabaster, AL).
  • Biotin-x-DPPE was purchased from Molecular Probes (Eugene, OR), sulforhodamine B was purchased from Eastman Chemical Co. (Rochester, NY), and Carnation non-fat dry milk powder (CNDM) was obtained locally.
  • Protein assay dye reagent, goat anti-rabbit IgG alkaline phosphatase conjugate and the substrates for alkaline phosphatase were purchased from Bio-Rad Laboratories (Hercules, CA).
  • Silica gel TLC flexible plates and preparatory silica gel plates, both containing fluorescent indicator, were obtained from Whatman (Maidstone, England).
  • Plastic-backed nitrocellulose membranes with pore sizes >3 ⁇ m were obtained from Schleicher & Schuell, Inc.
  • Antibodies to the immunogenic conjugation product were raised in two New Zealand white rabbits at the Cornell College of Veterinary Medicine.
  • the IgG fraction was purified from antisera by the caprylic acid/ammonium sulfate precipitation method, described in McKinney, M.M.; Parkinson, A. J . Immunol . Methods 1987, 96, 271-279. Antibodies obtained in this manner were used in all subsequent studies.
  • Liposomes were designed to be competitive markers in an immunoassay for polychlorinated biphenyls (PCB), and this was accomplished by incorporating previously coupled 2ClPB-DPPE into liposome bilayers by the reverse-phase evaporation method described in Siebert, T.A.; Reeves, S.G.; Durst, R.A. Anal . Chim . Acta 1993, 282, 297-305, from a mixture of DPPC, cholesterol, DPPG, 2ClPB-DPPE conjugate, and biotin-x-DPPE in a molar ratio of 5:5:0.5:0.1:0.01.
  • Nitrocellulose membrane was cut into 8 x 15 cm sheets, thoroughly wetted with 10% methanolic TBS, pH 7.0, and dried under vacuum. The membrane sheet was mounted on a Linomat IV (CAMAG Scientific Inc., Wrightville Beach, NC) microprocessor controlled TLC sample applicator. Solutions of anti-PCB antibodies (0.2 mg mL -1 ) and anti-biotin antibodies (0.5 mg mL -1 ) were applied at 1.25 ⁇ L s -1 , for 85 s, with 190 kPa (27.5 psi) N 2 producing anti-biotin zones as shown in Figure 8A and 8B and anti-PCB zones as shown in Figure 8A.
  • Linomat IV CAMAG Scientific Inc., Wrightville Beach, NC
  • the format for the LIC assay shown in Figure 8A, consists of a solution containing PCB-tagged liposomes and a nitrocellulose test strip with immobilized anti-PCB and anti-biotin capture zones in sequence.
  • the LIC assay is performed by dispensing 25 ⁇ L of the sample (in water or up to 30% methanol extraction solvent) and 2.5 ⁇ L of a 3 times concentrated solution of TBS buffer into a 10 x 75 mm glass test tube, mixing the contents, and adding 25 ⁇ L of a liposome solution (stock liposome solution diluted to 3.9 x 10 4 liposomes ⁇ L -1 ) .
  • the format for the LIA assay shown in Figure 8B, is modified by adding 30 picomoles of anti-PCB antibody/drop to the concentrated TBS solution. Furthermore, after mixing, the solution is allowed to incubate at room temperature for 15 minutes before continuing with the assay. After these initial preparations the test tube is shaken mildly to mix the contents and the test strip is inserted. In both formats, the strip is left in the tube until the solution front reaches the end of the strip (ca. 8 min); the strip is then removed and air dried. The color intensity of all zones is estimated visually or by scanning densitometry.
  • the LIC format has two immobilized reagent zones, shown in Figure 5A.
  • Buffered solutions containing liposomes and various amounts of PCBs migrate vertically through channels in the nitrocellulose polymer matrix by capillary action and sequentially pass through the competition and measurement zones.
  • Figure 5A depicts the competitive binding events that occur on the surface of the nitrocellulose during LIC. It should be emphasized that this reaction occurs in a flowing solution and is therefore under non-equilibrium conditions.
  • Initial experiments have shown that the ratio of liposome binding at the two zones remains constant despite changes in the liposome concentration over two orders of magnitude. Only the absolute signal intensity changed in these experiments.
  • the antibody competition zone will display an inverse relationship to the amount of analyte present.
  • a second zone of anti-biotin is applied, at a high enough concentration (11.9 picomoles/sensor), to capture all liposomes that escape binding in the competition zone at maximal inhibition from analyte. This zone displays a proportional relationship between color intensity and analyte concentration.
  • the LIA format consists of a homogeneous aggregation reaction, migration on a nitrocellulose sensor, and detection in an immobilized anti-biotin capture zone, as shown in Figure 5B.
  • All liposome particles must move through a tortuous path in the nitrocellulose matrix. In doing so, large particle sizes will tend to be retained in the interstices and will form the aggregation zone. Smaller particle sizes will not be inhibited and will continue to migrate until bound in the anti-biotin collection zone.
  • the bivalent IgG and multi-haptenated liposomes undergo multiple crosslinking events, as depicted in Figure 6A.
  • the resulting multi-liposome structures are similar to those reported for avidin/biotin induced liposome aggregation in Chiruvolu, S.; Walker, S.; Israelachvili, J.; Schmitt, J.-J.; Leckband, -D. ; Zasadzinski, J.A. Science 1994, 264, 1753-1756.
  • PCBs inhibit aggregation, and an inversely proportional signal will be displayed at the aggregation zone, which forms at the meniscus of the test solution in which the test device is inserted.
  • the liposomes that do not aggregate will be proportional to the amount of analyte in solution and bind to the measurement zone as described for the LIC sensor.
  • An advantage of the LIA format over the LIC format is that the competitive reaction is allowed to more closely approach equilibrium during the incubation period and thus achieves higher sensitivity. Furthermore, no analyte-specific antibody is immobilized on the nitrocellulose. With only the measurement zone remaining, the LIA strips become a generic immunoassay component that can be used with any suitable incubation reaction.
  • the two formats presented consist of an inversely proportional and a directly proportional zone where signal intensity can be measured.
  • the lower, inversely proportional zone of each type of sensor will be referred to here as the reaction or R zone because of its close relationship with the competitive binding events between liposomes and antibody.
  • the higher, directly proportional zone will be referred to as the collection or C zone, which indicates that this zone only serves to accumulate liposomes not retained in the reaction zone due to the presence of analyte.
  • Liposome stock solutions were stored at 4°C, in the dark, and were essentially 100% intact after 7 months of storage. This is in agreement with other reports which have also noted quite stable liposome preparations with storage at 4°C. Plant, A.L.; Brizgys, M.V.; Locascio-Brown, L.;-Durst, R.A. Anal Biochem 1989, 176, 420-426; Pinnaduwage, P.; Huang, L.
  • the characteristics of the liposomes prepared as described in Example 6 are listed in Table II.
  • the mean diameter is slightly smaller than reported previously, Siebert, T.A.; Reeves, S.G.; Durst, R.A. Anal. Chim. Acta 1993, 282, 297-305.
  • three significant changes have been made: the entrapped dye was increased two-fold to 200 mM, DPPE-biotin was incorporated, and the total amount of analyte-DPPE was increased ten-fold to 1 mole %.
  • These changes could account for the formation of liposomes with slightly different characteristics. See Liposomes: A Practical Approach; New, R.R.C., Ed.; Oxford University Press: New York, 1990, 301.
  • the proper concentration of liposomes to be used in the immunomigration assays was determined by establishing two initial criteria: in either format, the collection (i.e., measurement) zone (C) must provide a greyscale density reading of at least 7.500 (arbitrary units), and the ratio of the reaction zone (R) (competitive binding portion in LIC format; aggregation zone in LIA) to the capture zone (R/C) must be less than or equal to 1, at an analyte concentration that causes maximal inhibition.
  • the two criteria insure that the signal intensity will be visually obvious and that greater than 50% of the liposomes that could potentially accumulate in the R zone will be released to the C zone.
  • a liposome concentration of 1.3 x 10 4 liposomes ⁇ L -1 was observed to give a C-zone greyscale reading of 10.325 arbitrary units for the LIA and 8.916 for the LIC formats.
  • This liposome concentration also provided a R/C of 0.72 and 0.51 for the LIC and LIA, respectively, after addition of 10 nanomoles of Aroclor 1232. If the total signal is summed for the R and C zones and considered to present 100% of the liposomes available for detection, then the R/C ratio can be used to determine the percentage that is associated with each individual zone. Therefore, 58.2% and 66.2% of the liposomes, for LIC and LIA, respectively, were released to the C zone at maximal inhibition from Aroclor 1232. This satisfied our criteria and all subsequent experiments were carried out at this liposome concentration.
  • methanol differentially affects subpopulations of liposomes, then it should be possible to observe a change in the liposome size distribution upon exposure.
  • Table III lists the percentage of liposomes that are greater than a given particle size after exposure to methanol. For all particle sizes measured, exposure to an increasingly methanolic buffer results in an increase in the population percentage. This indicates that methanol exposure may preferentially lyse smaller diameter liposomes, i.e., ⁇ 0.5 ⁇ m, and thereby increase the relative percentage of larger diameter liposomes.
  • methanol may preferentially lyse smaller diameter liposomes, i.e., ⁇ 0.5 ⁇ m, and thereby increase the relative percentage of larger diameter liposomes.
  • increasing amounts of short-chain alcohols, including methanol induce a transition in DPPC from the bilayer gel to the interdigitated state.
  • the color intensities in the R and C zones in the LIA assay are indicators of the number of associations between liposomes and anti-PCB antibodies. With increasing concentrations of analyte, this association is increasingly inhibited and a lower color intensity will result in the R zone.
  • the color intensity observed in these zones is directly related to the binding strength between liposomes and antibody: if aggregation increases, then a higher binding affinity is indicated, as noted in Katoh, S.; Mori, Y.; Fujita, R.; Sada, E.; Kishimura, M.; Fukuda, H. Biotechnology and Bioengineering 1993, 41, 862-867.
  • the R and C zones are somewhat analogous to the measurements of antibody-bound and free analyte that are used in a Scatchard analysis for the determination of affinity constants for receptors and their ligands. See Scatchard, G. Ann. N.Y. Acad. Sci, 1949, 660-672. In a Scatchard analysis, the ratio bound/free analyte is plotted as a function of the amount of analyte bound, and the slope of this plot equals the affinity constant. The strip is, in essence, acting to separate those liposomes which have not been bound by antibody from those which have.
  • affinity constants The reason this device cannot be used for the exact determination of affinity constants is that the R zone intensity is actually produced by large aggregates resulting from numerous multiple binding events and likewise the C zone intensity is most probably produced by free liposomes and small aggregates. Thus a detailed Scatchard analysis using LIA is not able to provide an affinity (or avidity) constant in the traditional sense. However, the device can be used to compare relative antibody affinities and provide useful information for the selection of antibodies within the context of the liposome-based immunoassays.
  • Antisera from two different rabbits were analyzed for their liposome binding ability as a way to determine their suitability for use in the further development of immunomigration PCB assays. Thirty nanomoles of either antibody preparation (identified below as #1 and #2) were added to liposome solutions, without PCBs, and incubated for 15 minutes according to the standard LIA protocol. Antibody #1 displayed a R/C of 4.16 and antibody #2 displayed a R/C of 60.9. These ratios indicate that 81% and 98% of available liposomes, for antibody #1 and #2 respectively, are bound in the R zone. On this basis, antibody #2 was chosen for its high R/C ratio which indicates its greater ability to induce immunoaggregation of PCB-tagged liposomes. Furthermore, the small C zone signal produced with antibody #2 was desired in order to minimize the background signal for blank solutions.
  • Varying amounts of antibody #2 were tested using LIA in order to determine the optimal amount of antibody to be used during the incubation step.
  • LIA was performed with solutions containing zero and 27 picomoles of Aroclor 1232.
  • the greyscale density at the C zone was measured and is displayed in Figure 8.
  • Antibody amounts lower than 20 picomoles induced very little aggregation and therefore only a small signal change was observed with the addition of PCBs.
  • aggregation was more complete but, again, only a small signal change was observed upon addition of PCBs.
  • These levels present too many PCB binding sites and can readily bind both analyte and PCB-tagged liposomes without inhibiting immunoaggregation.
  • Thirty nanomoles of antibody #2 displayed the largest signal difference between the blank and PCB solutions and, therefore, was used for all subsequent experiments.
  • liposome aggregation Several forces can act to induce liposome aggregation, including van der Waals, ion-binding, hydrophobic interactions, depletion forces, and receptor-ligand interactions, as described in Chiruvolu, S.; Walker, S.; Israelachvili, J.; Schmitt, J.-J.; Leckband, D.; Zasadzinski, J . A . Science 1994, 264, 1753-1756.
  • the receptor-ligand interactions are the only forces that can be considered to induce specific aggregation since they only act at specific points on the liposome surface and only when the interacting molecules are present.
  • the time-dependent development of liposome-antibody aggregates was investigated using laser diffraction particle size analysis and is shown in Figure 9.
  • the liposome size distribution continues to change with time and all distributions pass through a point at 0.66 ⁇ m. All of the size distributions decrease essentially to zero % above a diameter of two microns.
  • Obscuration is a measurement of solution turbidity and will increase with an immunoaggregation induced increase in particles sizes. See Collord, B.C.; Marchal, E.; Humbert, G.; Linden, G.; Montagne, P.; El-Bari, N.; Duheille, J.; Varcin, P.J. Dairy Sci 1991, 74, 3695-3701. Sample obscuration is especially useful for comparing liposomes of identical composition and concentration for relative changes in the entire size distribution. See Liposomes: A Practical Approach; New, R.R.C., Ed.; Oxford University Press: New York, 1990, 301.
  • Figure 10A illustrates the time-dependent increase in laser light obscuration observed for liposome solutions containing 0 and 100 ppb of Aroclor 1232.
  • the 100.ppb solutions correspond to 27 picomoles of Aroclor 1232 per test.
  • Both solutions aggregated following logarithmic trends which demonstrates that, while both solutions continue to increase in their mean size, the aggregation begins very rapidly.
  • This study also shows that a significant inhibition of liposome immunoaggregation with 100 ppb of PCBs reaches a maximum between 5 and 20 minutes.
  • More information may be obtained from analysis of all particle information at each time point, as shown in Figure 10B.
  • the z axis is the observed percentage difference in the liposome size distribution with the blank solution being subtracted from the 100 ppb PCB solution. Therefore, in this analysis, positive percentage differences indicate a greater number percentage in the PCB solution and negative differences indicate a greater percentage in the blank solution.
  • An increasing percentage difference in small diameter particles is the most obvious trend, indicated by the tall peak located at the very smallest ( ⁇ 0.66 ⁇ m) liposome diameters.
  • PCB-containing solutions will retain a large percentage of liposomes of small diameters and would be expected to contribute to a higher C zone signal.
  • increases in PCB concentration causes a corresponding increase in the observed C zone signal.
  • the second trend to be noted is the trough formed at diameters between 0.66 and 2 ⁇ m, and which has a mode of 1 ⁇ m. This trough begins around 5 minutes and is level at about 15 minutes. This trend indicates that particles of this subgroup increase with time in the blank over the PCB-containing solutions. These data correlate well with the obscuration data as no significant difference can be seen before 5 minutes, and that the difference is essentially level by 15 minutes.
  • the particle size range composing the trough is expected to contribute to the high R zone signal that is observed with blank solutions.
  • gelatin blocking was chosen for its ability to allow fast liposome migration and yet maintain a nonspecific background signal close to the ideal nitrocellulose surface to which no liposomes have been applied. Concentrations of gelatin lower than 0.25% in the blocking solution were not expected to significantly improve the assay as solvent migration time was already equal to a non-blocked strip. Effect of gelatin blocking on non- specific liposome binding and overall assay migration time. % Gelatin in Blocking Solution Background Signal O.D. Migration Time Minutes 0.00 2.533 7.75 0.25 3.253 7.72 0.50 3.678 8.15 0.75 3.999 9.93
  • Table V displays these values and calibration curve equations for both the LIC and LIA formats. All values on the calibration curves fell very close to a true logarithmic trend, as evidenced by the high correlation coefficients and that the relative standard deviations for calibration points were approximately 6% for both assays.
  • the calibration curve for the LIC assay was read at the inversely proportional R zone.
  • One possible reason for the lack of response at the LIC C zone could be the retention of liposomes by the membrane nonspecific binding sites resulting from the increased migration distance, as shown in Figure 5A.
  • the LIC will give a positive result for amounts of Aroclor 1232 amounts greater than 0.4 nanomoles.
  • the LIC format is not intended to be used quantitatively but the 99% C.I. is still listed for illustrative purposes. It can be seen in Table V that the LIA assay will detect PCBs at levels more than two orders of magnitude less than the LIC assay.
  • the slope of the calibration curve indicates that the sensitivity of the LIA assay is approximately 2.6 times greater than the LIC assay. Associated with an increased sensitivity are increased signal and dynamic ranges for LIA. These ranges indicate the signal differences between zero and maximum analyte values.
  • the LIA assay will be the method of choice.
  • Other commercially available ELISA test provide standardized color cards for use if quantitation is desired and a similar scheme could be used for the LIA format.
  • the advantage of the LIC format is that the entire assay takes less than 8 minutes because it does not require the aggregation step.
  • the main advantage of LIC assays would come in situations when only threshold (e.g., tolerance level) measurements are required (>0.4 nanomoles/test) and when analysis time is critical.
  • threshold e.g., tolerance level
  • the anti-PCB antibodies used in this study were expected to more readily bind to lower chlorinated mixtures due to the choice of a monochlorinated hapten for antibody production.
  • Table VI shows the relative binding affinities of several Aroclors spanning the range of chlorine content that was present in commercial mixtures.
  • the 2-chlorobiphenyl (2ClBP) which is analogous to our hapten was tested and assigned a value of 100% due to its similar structure to the hapten.
  • the percent reactivity was calculated by subtracting the background from all C zone greyscale readings and then comparing the various Aroclor mixtures to 2ClBP. All aroclors were tested at 100 ppb and could be detected using the polyclonal antibodies.
  • Both of these devices are designed as single-use immunosensors which will reduce the problems of fouling and cross-contamination which are especially prevalent in field screening applications. All reagents are produced using standard techniques and are stable at room temperature. The devices are fabricated from commercially available nitrocellulose membranes. Furthermore, the LIA sensors do not contain an immobilized anti-analyte zone and thus can be used generically for any test for which a suitable incubation reaction has been developed.
  • the LIC device has been proposed as a rapid screening device for the detection of PCBs with amounts greater than 0.4 nanomoles per test.
  • the LIA device which is based on the size separation of liposome aggregates, is intended for more sensitive and quantitative measurements but requires an additional 15-minute incubation step..
  • the LIA assay can detect PCB amounts down to 2.6 picomoles in ca. 23 minutes.
  • the incubation step for the LIA assay was performed with only an initial manual mixing of reagents and with a single antibody reagent. This was done to maintain the simplicity of the prototype device.
  • Others have noted that particle aggregation immunoassays often attain enhanced efficiency with the addition of a second anti-IgG antibody. See Monroe, D. J. Liposome Res.
  • Table VII is a comparison of assay performance for two analytes (PCB and Alachlor) in the method of the invention.
  • the data for Alachlor were obtained by procedures substantially as described in Examples 3-9 above. However, a greater (approximately 10-fold) concentration of PVP was employed in the blocking reagent, and pH-perturbed antibodies were employed.
  • the assay methodology of the invention could potentially be applied to multianalyte environmental detection.
  • Another major advantage of the use of a liposome reagent is their unique ability to entrap an almost unlimited range of colored, fluorescent, luminescent, and electroactive signal markers which can be used for multianalyte testing.
  • Immunoassays which do not employ liposomes but which can simultaneously detect more than one analyte by incorporating separate labels or spatially resolving analyte binding have been described in Hayes, F.J.; Halsall, B.; Heineman, W. Anal. Chem.
  • Liposome-based immunoassays provide an excellent means of delivering multiple labels and for instantaneously releasing ca. 10 7 label molecules per binding event.
  • test device packaged as part of a commercial kit might include special holders for individual strips, in which openings are provided for sample application and optical readout, for example, as shown in Figure 4.
  • the strips could be run in almost any orientation, e.g., lateral, instead of vertical flow.
  • a dual-strip test device as shown in Figure 2 may be employed.
  • the strip is divided in two by removal of a very thin strip of nitrocellulose from the plastic backing, thereby providing two identical strips with a hydrophobic separation to prevent solution cross-talk.
  • a tolerance level control is applied to the strip adjacent to the sample, and both are run simultaneously. This verifies the strip performance and provides a more quantitative interpretation of the strip results.
  • multi-analyte assays may also be conducted using the test device and method of the invention.
  • the use of immunospecific liposome migration offers many advantages for the extra-laboratory detection of environmental contaminants. Devices utilizing this technique are fast, easy to use, robust, and respond to the presence of analyte at low-parts-per billion concentrations.
  • Two complementary prototype liposome-based immunomigration techniques have been developed for the detection of polychlorinated biphenyls (PCBs).
  • PCBs polychlorinated biphenyls
  • the liposome immunocompetition (LIC) assay format measures the competitive reaction between analyte-tagged liposomes and the sample analyte for immobilized antibodies and can detect 0.4 nanomoles of PCB in less than 8 minutes.
  • the liposome immunoaggregation (LIA) assay detects the inhibition of immunospecific liposome aggregation in solution and can detect 2.6 picomoles of PCB in less than 23 minutes.
  • Laser diffraction particle sizing has been used to study LIA induced increases in liposome size over time and to determine optimal conditions for the application of this technique.
  • Both formats utilize capillary action to transport liposome-containing solutions along strips of nitrocellulose. Measurement of color intensity is then carried out visually or with a desktop scanner.
  • the liposome immunocompetition (LIC) format has been previously investigated for detection of the herbicide, alachlor, as described in Siebert, T.A.; Reeves, S.G.; Durst, R.A. Anal. Chim. Acta 1993, 282, 297-305, and is now shown to be effective for the detection of PCBs.
  • the present sensor format is based on the principle of immunoaggregation (liposome immunoaggregation or LIA) between anti-PCB antibodies and analyte-tagged liposomes.
  • LIA liposome immunoaggregation
  • This second device represents a novel use of immunoaggregation for the detection of PCBs with a field-portable device. Both of these PCB sensors are fast, simple to use, and can be evaluated visually.

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Claims (19)

  1. Procédé pour détecter ou quantifier un analyte dans un échantillon à tester, comprenant :
    fournir un dispositif de test comprenant un matériau absorbant, lequel matériau absorbant comprend :
    une partie de contact au niveau ou à proximité d'une première extrémité dudit matériau absorbant ; et
    une partie de mesure à un endroit dudit matériau absorbant qui est situé à distance de la première extrémité, ladite partie de mesure ayant un récepteur pour un conjugué d'un analogue de l'analyte et des liposomes, dans laquelle lesdits liposomes comprennent un marqueur détectable ;
    combiner une matière de liaison spécifique de l'analyte avec le conjugué et l'échantillon à tester pour former un mélange ;
    incuber le mélange pendant une durée suffisante pour permettre la compétition entre tout analyte présent dans l'échantillon à tester et le conjugué pour la matière de liaison ;
    mettre en contact le mélange avec ladite partie de contact dudit matériau absorbant après ladite incubation ;
    laisser le mélange migrer, à partir de la partie de contact, dans ladite partie de mesure dudit matériau absorbant ;
    détecter la présence ou une quantité dudit marqueur dans ladite partie de mesure dudit matériau absorbant ; et
    corréler la présence ou une quantité dudit marqueur dans ladite partie de mesure dudit matériau absorbant respectivement avec la présence ou une quantité de l'analyte dans l'échantillon.
  2. Procédé selon la revendication 1, dans lequel l'analogue de l'analyte est soit l'analyte, soit un analogue réactif de l'analyte.
  3. Procédé selon la revendication 1, dans lequel ladite mise en contact est réalisée en introduisant la partie de contact du matériau absorbant dans le mélange.
  4. Procédé selon la revendication 1, dans lequel la partie de contact comprend en outre une partie à effet capillaire au niveau de la première extrémité, ladite mise en contact étant réalisée en déposant une goutte du mélange sur le matériau absorbant dans la partie de contact hors de la zone à effet capillaire, et ladite étape consistant à laisser le mélange migrer comprenant l'introduction de la partie à effet capillaire dans un réactif à effet capillaire.
  5. Procédé selon la revendication 1, dans lequel ladite mise en corrélation est utilisée pour déterminer la quantité d'analyte dans l'échantillon à tester et est réalisée en comparant la quantité de marqueur dans la partie de mesure avec un ou plusieurs étalons de référence ayant des concentrations connues de l'analyte pour des quantités particulières de marqueur, afin de déterminer la concentration de l'analyte dans l'échantillon à tester par rapport aux concentrations connues.
  6. Procédé selon la revendication 1, dans lequel le marqueur est un colorant fluorescent, un colorant visible, une matière bioluminescente, une matière chimioluminescente, une matière radioactive ou un substrat enzymatique.
  7. Procédé selon la revendication 6, dans lequel le marqueur est un colorant fluorescent ou un colorant visible et ladite détection est réalisée par une mesure fluorométrique ou spectrophotométrique.
  8. Procédé selon la revendication 6, dans lequel le marqueur est un colorant visible et ladite détection est réalisée visuellement à l'oeil nu.
  9. Procédé selon la revendication 1, dans lequel les liposomes sont préparés à partir d'un ou plusieurs phospholipides, glycolipides, stéroïdes, alkylphosphates ou esters d'acides gras.
  10. Procédé selon la revendication 9, dans lequel les phospholipides sont choisis dans le groupe constitué de la lécithine, de la sphingomyéline et du dipalmitoyle, et les stéroïdes sont choisis dans le groupe constitué du cholestérol, du chlorestanol et du lanostérol.
  11. Procédé selon la revendication 1, dans lequel les liposomes sont préparés à partir d'un mélange comprenant de la dipalmitoylphosphatidylcholine, du cholestérol et du dipalmitoylphosphatidylglycérol.
  12. Procédé selon la revendication 1, dans lequel l'analyte est un antigène ou un haptène, et la matière de liaison est un anticorps reconnaissant l'antigène ou l'haptène.
  13. Procédé selon la revendication 1, dans lequel l'analyte est l'alachlor, un PCB, la digoxine, une hormone, une vitamine, un métabolite ou un agent pharmacologique.
  14. Procédé selon la revendication 1, dans lequel le récepteur est capable de se lier soit aux liposomes, soit à un composant de surface des liposomes.
  15. Procédé selon la revendication 14, dans lequel le composant de surface et le récepteur sont chacun choisis dans le groupe constitué de l'avidine, de la biotine et de l'anti-biotine.
  16. Procédé selon la revendication 1, dans lequel ladite détection comprend la lyse desdits liposomes pour libérer ledit marqueur.
  17. Procédé selon la revendication 1, dans lequel ledit matériau absorbant comprend en outre une région pour l'accumulation d'agrégats formés à partir dudit conjugué et de ladite matière de liaison, dans lequel ladite région pour l'accumulation est située à distance de ladite partie de mesure et soit entre ladite partie de mesure et ladite partie de contact, soit dans ladite partie de contact.
  18. Procédé selon la revendication 1, dans lequel ledit récepteur est lié à ladite partie de mesure.
  19. Procédé selon la revendication 1, dans lequel ledit marqueur est encapsulé dans lesdits liposomes.
EP96909480A 1995-02-01 1996-01-29 Dispositif de dosage et de test pour l'immunoagregation amelioree par des liposomes Expired - Lifetime EP0807255B1 (fr)

Applications Claiming Priority (3)

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US382482 1995-02-01
US08/382,482 US5756362A (en) 1993-10-12 1995-02-01 Liposome-enhanced immunoaggregation assay and test device
PCT/US1996/001617 WO1996024062A1 (fr) 1995-02-01 1996-01-29 Dispositif de dosage et de test pour l'immunoagregation amelioree par des liposomes

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EP0807255A1 EP0807255A1 (fr) 1997-11-19
EP0807255A4 EP0807255A4 (fr) 2000-05-24
EP0807255B1 true EP0807255B1 (fr) 2005-12-21

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EP (1) EP0807255B1 (fr)
AU (1) AU707803B2 (fr)
CA (1) CA2211132C (fr)
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DE69635607D1 (de) 2006-01-26
AU5295996A (en) 1996-08-21
EP0807255A4 (fr) 2000-05-24
EP0807255A1 (fr) 1997-11-19
DE69635607T2 (de) 2006-09-14
US6086748A (en) 2000-07-11
AU707803B2 (en) 1999-07-22
US5753519A (en) 1998-05-19
WO1996024062A1 (fr) 1996-08-08
CA2211132C (fr) 2008-10-07
US5756362A (en) 1998-05-26
CA2211132A1 (fr) 1996-08-08

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